Selection of a human anti-progesterone antibody fragment from a transgenicmouse library by ARM ribosome display

In antibody-ribosome-mRNA complex (ARM) ribosome display, stable complexes of nascent protein, mRNA and ribosomes are produced in a eukaryotic in vitr o expression system, through coupled transcription and translation of DNA l acking a 3' stop codon. Selection of the protein simultaneously captures th e relevant mRNA, which is recovered as DNA by coupled reverse transcription -polymerase chain reaction (RT-PCR) performed on the intact complexes. Here , we describe the use of ARM display to select a specific human antibody fr agment from a transgenic mouse library. The mice carry unrearranged gene se gments of the human heavy (H) and kappa light (L) chain loci, while the end ogenous murine H and kappa loci are functionally silenced; they respond to immunisation by production of fully human IgM antibodies. A library encodin g human single-chain (sc) antibody (V-H/K) fragments, in which V-H domains and kappa light chains were combined at random by PCR, was prepared from sp leen cells of transgenic mice immunised with progesterone-bovine serum albu min (BSA). Library diversity was demonstrated by sequencing. Progesterone-b inding fragments were selected over five cycles of ARM display and the sele cted DNA cloned and expressed in Escherichia coli. Soluble V-H/K fragments obtained in periplasmic extracts had the same specificity as ribosome-bound V-H/K, supporting the view that folding and specificity of the displayed a nd soluble proteins are equivalent. The affinity of the expressed V-H/K was similar to 10(-8) M. Sequencing showed that ARM display selected a single V-H/V-L combination (V(H)1-2, V kappa 4-1) and rearrangement, with a few mu tational differences between clones. Monoclonal antibodies against progeste rone-BSA obtained from hybridomas were encoded by the same V-H and V-L segm ents and had similar properties to the fragments obtained in vitro. The com bination of ribosome display and transgenic mouse technologies is a rapid m eans of generating fully human antibody fragments in vitro for expression a nd further manipulation. (C) 1999 Elsevier Science B.V. All rights reserved .